Hemostasis or stoppage of bleeding involves the interplay of two biochemical pathways (the extrinsic and intrinsic pathways) which are controlled by various protein factors and formed elements, e.g., platelets. The processes by which blood coagulates as it is presently understood involve a multi-step cascade of activations of the protein factors that culminate in fibrin formation. Interference with any one step in these intricate processes hinders proper blood clotting and can result in significant bleeding.
Various tests have been developed to test the individual steps of the cascade involved in blood clotting in order to determine whether the blood of a patient can properly clot or whether there is clotting disorder in which there is a deficiency of one or more of the factors necessary for proper clotting.
There are a number of bleeding disorders which result in abnormal blot clotting including von Willebrand disease, factor VIII deficiency (hemophilia), afirinogenimia, platelet dysfunction (e.g., Bernard-Soutier Syndrome, Glanzman's Thrombasthemia, and storage pool disease). Individuals afflicted with these disorders suffer a potential risk of severe bleeding. Methods to determine the deficiency which is the cause of the disorder are desirable for immediate clinical and therapeutic intervention.
It is well known that the condition of the platelets or the platelet function of blood is one indication of the ability of blood to properly clot. vWf deficiency is one of the more common types of platelet dysfunction that occurs in approximately 1% of the population. vWf is a large multimeric glycoprotein which has an important role in the adhesion of platelets to the subendothelium following blood vessel damage. The interaction of vWf is essential for normal hemostasis.
The more common types of von Willebrand disease (vWD) include: a lack of the normal amount of vWf (mild or severe deficiency), and type IIA and type IIB vWD (which result from the deficiency of functional vWf proteins). Type IIB vWD is characterized by the absence of the high molecular weight multimers of vWf in the plasma. Type IIA vWD is characterized by the absence of both the intermediate and the high molecular weight multimers of vWf. Type IIA is further characterized by a decreased affinity of vWf for the platelet receptor glycoprotein Ib (GPIb), whereas in type IIB vWD, the vWf has an increased affinity for GPIB. See Ribba, et al., J. Biol. Chem., 267 (32):23209-15 (1992). Type IIA is the most common type II variant of vWD. Ruggeri, et al., J. Clin. Invest., 65:1318 (1980).
The primary existing test in use for testing platelet function or primary hemostasis on patients is known as the "bleeding time test". The bleeding time test which has existed for several decades involves making an incision in the forearm of the patient with a blood pressure cuff inflated to 40 mm Hg. Filter-paper is used to absorb the blood from the incision and to determine the amount of time for bleeding to stop. Bleeding usually stops within 10 minutes. The clinical utility of the test is limited by variability associated with the depth of the incision, the pressure applied, fluctuations in blood pressure of agitated patients, the method of absorbing blood with the filter paper, the direction of the cuts, among others. The variables are difficult to control and lead to problems with standardization and interpretation. Accordingly, a test for platelet function which does not involve making an incision and which is also more accurate was developed.
U.S. Pat. Nos. 4,604,894; 4,780,418; and 5,051,239 disclose an assay system which can be used to perform an in vitro test on blood for platelet function, the results of which can be correlated to the in vivo bleeding time test described above, thereby eliminating involvement of the patient. The Thrombostat.TM. 4000 (Dade International, Inc.), in current use, is one such system.
Platelet function is evaluated in the Thrombostat.TM. 4000 by aspirating anticoagulated whole blood samples at a constant negative pressure through a small aperture positioned at the center of a separating wall which may be non-porous or porous. In systems wherein the separating wall is porous, it is wetted prior to the start of the assay with an activator that activates coagulation of blood platelets. A platelet plug forms at the aperture and the time required for the cessation of blood flow to occur is determined. The time required to obtain full occlusion of the aperture is termed "in vitro bleeding time". This time is then correlated to platelet function assessed by conventional platelet aggregometry, or in vivo bleeding time.
The aforementioned in vitro assay systems which enable the determination of platelet dysfunction, however, do not discriminate between the various deficiencies which can cause the abnormal clotting. A physician utilizing such a system therefore does not know whether the dysfunction is caused by a vWf deficiency, or a less common platelet function disorder, thereby making treatment more difficult.
The current techniques for indicating vWf deficiency include: quantitative assays, e.g., immunologic detection of vWf; platelet aggregation assays, e.g. ristocetin cofactor assay with fixed platelets; and optical measurement of ristocetin-induced platelet aggregation. See Hemostasis and Thrombosis; Basic Principles and Clinical Practice, 3rd ed., eds. Colman, R. W., et al., J.B. Lippincott Co. (1994). These are static assays, in that they do not simulate in vivo clotting conditions. These assays are also performed on plasma, rather than whole blood, which adds steps and time to the performance of the assay. In some assays, platelet-rich plasma is used, other assays use platelet-poor plasma with added platelets, while still others use washed, gel-filtered or fixed platelets. See eg., Miller, et al., J. Clin. Invest., 72:1532-1542 (1983); Allain, et al., J. Lab. Clin. Med., Feb.:318-328 (1975).
It would be useful to have a von Willebrand factor deficiency assay which tests clotting on samples of whole blood under conditions that are representative of in vivo conditions. The currently known techniques, described above, are complicated, time consuming and available only in specialized clinical laboratories. Results are usually delayed, sometimes requiring half a day under the best of circumstances, and more typically requiring 1 to 2 days in the usual clinical setting. This delay in obtaining the results leads to a delay in identification of the cause of the bleeding disorder and a delay in treatment. Delayed results are unacceptable in certain situations, such as in emergency surgical procedures.
It is therefore desirable to have a method for rapid indication of vWf deficiency.